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CA2358326A1 - Method for separating cells using discontinuous density gradient centrifugation - Google Patents

Method for separating cells using discontinuous density gradient centrifugation Download PDF

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Publication number
CA2358326A1
CA2358326A1 CA002358326A CA2358326A CA2358326A1 CA 2358326 A1 CA2358326 A1 CA 2358326A1 CA 002358326 A CA002358326 A CA 002358326A CA 2358326 A CA2358326 A CA 2358326A CA 2358326 A1 CA2358326 A1 CA 2358326A1
Authority
CA
Canada
Prior art keywords
cells
particles
density
dense particles
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002358326A
Other languages
French (fr)
Inventor
Steven M. Woodside
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
StemCell Technologies Inc
Original Assignee
StemCell Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by StemCell Technologies Inc filed Critical StemCell Technologies Inc
Priority to CA002358326A priority Critical patent/CA2358326A1/en
Priority to CA2405881A priority patent/CA2405881C/en
Publication of CA2358326A1 publication Critical patent/CA2358326A1/en
Abandoned legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3616—Batch-type treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3693—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits using separation based on different densities of components, e.g. centrifuging
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/10—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by centrifugation ; Cyclones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0427—Platelets; Thrombocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2202/00—Special media to be introduced, removed or treated
    • A61M2202/04—Liquids
    • A61M2202/0413—Blood
    • A61M2202/0439—White blood cells; Leucocytes
    • A61M2202/0443—Macrophages, e.g. monocytes

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Vascular Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Anesthesiology (AREA)
  • Cardiology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Ecology (AREA)
  • Biophysics (AREA)
  • Sustainable Development (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention is related to a method for separating desired cells from undesired cells in a sample by discontinuous density gradient separation using dense particles to target unwanted cells and a density separation medium (DSM) that is at least about 0.001 g/cm3 higher than the density of the desired cells.

Claims (26)

1. A method for separating desired cells from undesired cells in a sample comprising:
- linking dense particles to the undesired cells in the sample to increase the effective density of the undesired cells;
- layering the sample over a density separation medium (DSM) having a density at least about 0.001 g/cm3 greater than the mean density of the desired cells;
- centrifuging the sample and DSM; and - recovering the desired cells from the interface between the DSM
and the sample.
2. The method according to claim 1 wherein the dense particles are selected from the group consisting of red blood cells, silica particles, metal particles, polymer particles, glass particles.
3. The method according to any of claims 1-2, wherein the dense particles are selected from the group consisting of red blood cells and silica particles.
4. The method according to any of claims 1-3, wherein the osmolarity of the DSM is approximately the same as the osmolarity of the sample
5. The method according to claim 4, wherein the osmolarity of the DSM is about 270 to about 300 mOsm.
6. The method according to any of claims 1-5, wherein the undesired cells are linked to the dense particles by drug-drug receptor, antibody-antigen, hormone-hormone receptor, growth factor-growth factor receptor, carbohydrate-lectin, nucleic acid sequence-complementary nucleic acid sequence, enzyme-cofactor or enzyme-inhibitor binding.
7. The method according to an of claims 1-6, wherein the undesired cells are defined by specific surface proteins and the dense particles are linked to these cells by antibodies specific for the cell surface proteins.
8. A method for separating desired cells from undesired cells in a sample comprising:
linking dense particles to the desired cells in the sample to increase the effective density of the desired cells;
- layering the sample over a density separation medium (DSM) having a density at least about 0.001 g/cm3 greater than the mean density of the undesired cells;
- centrifuging the sample and DSM;
- recovering the desired cells linked to the dense particles from the bottom of the sample; and - cleaving the dense particles from the desired cells.
9. The method according to claim 8, wherein the dense particles are selected from the group consisting of red blood cells, silica particles, metal particles, polymer particles, glass particles.
10. The method according to any of claims 8-9, wherein the dense particles are selected from the group consisting of red blood cells and silica particles.
11. The method according to any of claims 8-10, wherein the osmolarity of the DSM is approximately the same as the osmolarity of the sample
12. The method according to claim 11, wherein the osmolarity of the DSM
is about 270 to about 300 mOsm.
13 The method according to any of claims 8-12, wherein the undesired cells are linked to the dense particles by drug-drug receptor, antibody-antigen, hormone-hormone receptor, growth factor-growth factor receptor, carbohydrate-lectin, nucleic acid sequence-complementary nucleic acid sequence, enzyme-cofactor or enzyme-inhibitor binding.
14. The method according to an of claims 8-13, wherein the undesired cells are defined by specific surface proteins and the dense particles are linked to these cells by antibodies specific for the cell surface proteins.
15. A kit for performing the method according to any of claims 1-14 comprising one or more aliquots of a density separation medium having a density at least about 0.001 g/cm3 higher than the mean density of the desired cells, one or more aliquots of cell-specific targeting agents and one or more aliquots of dense particles.
16. The kit according to claim 15, wherein the dense particles are red blood cells and the cell-specific targeting agents are tetrameric antibody complex cocktails that link the undesired cells to the red blood cells.
17 The kit according to claim 15, wherein the cell-specific targeting agent is an antibody cocktail that binds the undesired cells and the dense particles bind to the undesired cells through interactions with the antibodies in the cocktail.
18. A kit for performing the method according to any of claims 1-14 comprising one or more aliquots of a density separation medium having a density at least about 0.001 g/cm3 higher than the mean density of the desired cells, and one or more aliquots of dense particles coated with one or more cell-specific binding agents that bind the undesired cells.
19. The kit according to any of claims 17-18, wherein the dense particles are silica particles.
20. The kit according to claim 19, wherein the dense particles are coated with one or more antibodies that bind the undesired cells.
21. A kit for performing the method according to any of claims 1-14 comprising one or more aliquots of a density separation medium having a density at least about 0.001 g/cm3 higher than the mean density of the undesired cells, one or more aliquots of cell-specific targeting agents and one or more aliquots of dense particles.
22. The kit according to claim 21, wherein the dense particles are red blood cells and the cell-specific targeting agent is a tetrameric antibody complex that links the desired cells to the red blood cells.
23. The kit according to claim 22, wherein the cell-specific targeting agent is an antibody that binds the desired cells and the dense particles bind to the desired cells through interactions with the antibody.
24. A kit for performing the method according to any of claims 1-14 comprising one or more aliquots of a density separation medium having a density at least about 0.001 g/cm3 higher than the mean density of the undesired cells, and one or more aliquots of dense particles coated with one or more cell-specific binding agents that bind the desired cells.
25. The kit according to any of claims 23-24, wherein the dense particles are silica particles.
26. The kit according to claim 15, wherein the dense particles are coated with one or more antibodies that bind the desired cells.
26. The kit according any of claims 16-26, further comprising printed instructions.
CA002358326A 2001-10-01 2001-10-01 Method for separating cells using discontinuous density gradient centrifugation Abandoned CA2358326A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002358326A CA2358326A1 (en) 2001-10-01 2001-10-01 Method for separating cells using discontinuous density gradient centrifugation
CA2405881A CA2405881C (en) 2001-10-01 2002-10-01 Method for separating cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CA002358326A CA2358326A1 (en) 2001-10-01 2001-10-01 Method for separating cells using discontinuous density gradient centrifugation

Publications (1)

Publication Number Publication Date
CA2358326A1 true CA2358326A1 (en) 2003-04-01

Family

ID=4170182

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002358326A Abandoned CA2358326A1 (en) 2001-10-01 2001-10-01 Method for separating cells using discontinuous density gradient centrifugation

Country Status (1)

Country Link
CA (1) CA2358326A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012024691A1 (en) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Multiphase systems for analysis of solid materials
EP2584360A1 (en) * 2007-12-05 2013-04-24 Zyomyx Inc. Cell assay kit and method
WO2019010186A1 (en) * 2017-07-03 2019-01-10 Trustees Of Tufts College Aqueous two-phase system for the separation and recovery of mammalian cells from contaminated cultures
EP3440193A4 (en) * 2016-04-07 2020-03-04 Mesotex, Inc. Process for isolating nucleated cells and nucleated cell populations and uses thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2584360A1 (en) * 2007-12-05 2013-04-24 Zyomyx Inc. Cell assay kit and method
WO2012024691A1 (en) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Multiphase systems for analysis of solid materials
WO2012024693A1 (en) * 2010-08-20 2012-02-23 President And Fellows Of Harvard College Density-based separation of biological analytes using multiphase systems
US9176105B2 (en) 2010-08-20 2015-11-03 President And Fellows Of Harvard College Density-based separation of biological analytes using multiphase systems
US9714934B2 (en) 2010-08-20 2017-07-25 President And Fellows Of Harvard College Multiphase systems and uses thereof
US9857353B2 (en) 2010-08-20 2018-01-02 President And Fellows Of Harvard College Kit for density-based separation of biological analytes using multiphase systems
US10436768B2 (en) 2010-08-20 2019-10-08 President And Fellows Of Harvard College Density-based separation of biological analytes using mutliphase systems
US10732167B2 (en) 2010-08-20 2020-08-04 President And Fellows Of Harvard College Multiphase systems and uses thereof
EP3440193A4 (en) * 2016-04-07 2020-03-04 Mesotex, Inc. Process for isolating nucleated cells and nucleated cell populations and uses thereof
WO2019010186A1 (en) * 2017-07-03 2019-01-10 Trustees Of Tufts College Aqueous two-phase system for the separation and recovery of mammalian cells from contaminated cultures
US12115471B2 (en) 2017-07-03 2024-10-15 Trustees Of Tufts College Aqueous two-phase system for the separation and recovery of mammalian cells from contaminated cultures

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